Superporous agarose--reticulated vitreous carbon electrodes for electrochemical sandwich bioassays.
We report on the use of flow-through electrodes fabricated from a composite of superporous agarose (SPA) and reticulated vitreous carbon (RVC) for carrying out sandwich bioassays via a model sandwich assay scheme. The flow-through design of the SPA-RVC electrodes allows for ease in solution handling with the use of micropipettors while allowing sandwich assays to be performed on the SPA matrix inside the RVC. A sandwich bioassay was devised for detecting biotinylated bovine serum albumin (b-BSA) as a proof-of-concept scheme to demonstrate applicability of SPA-RVC electrodes to carry out sandwich assays. In this bioassay scheme, SPA-RVC electrodes with avidin molecules immobilized on the SPA matrix were incubated with low quantities of b-BSA followed by incubation with avidinylated alkaline phosphatase (av-ALP). This construct creates a sandwich bioassay whereby b-BSA is sandwiched between the two avidin complexes. Av-ALP labels captured on the bound b-BSA catalytically hydrolyze conversion of 4-aminophenylphosphate (PAPP) to electrochemically active 4-aminophenol (PAP) which is then voltammetrically detected inside the RVC. The lower concentration detection limit for b-BSA was 0.32+/-0.1 ng mL(-1) and the absolute detection limit was 32+/-10 pg. Non-specific binding of av-ALP enzyme labels onto the avidin-activated SPA-RVC electrodes was low. Catalytic generation of PAP by non-specifically bound av-ALP occurs at a rate less than 2% of that for PAP generation by av-ALP in [(SPA-av)-(b-BSA-b)-(av-ALP)] sandwich constructs.
Rao
Ashwin K
AK
Advanced Material Research Laboratories, 91 Technology Drive, Anderson, SC 29625, USA.
Creager
Stephen E
SE
D000469:Alkaline Phosphatase / Q000737:chemistry; D001360:Avidin / Q000737:chemistry; D001681:Biological Assay / Q000295:instrumentation*; D002244:Carbon / Q000737:chemistry*; D004563:Electrochemistry; D004566:Electrodes; D004800:Enzymes, Immobilized / Q000737:chemistry; D016062:Porosity; D012680:Sensitivity and Specificity; D012685:Sepharose / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000032:analysis
D004800:Enzymes, Immobilized; D001360:Avidin; D012710:Serum Albumin, Bovine; D002244:Carbon; D012685:Sepharose; D000469:Alkaline Phosphatase